Adaptive introgression from maize has facilitated the establishment of teosinte as a noxious weed in Europe

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Adaptive introgression from maize has facilitated the

establishment of teosinte as a noxious weed in Europe

Valérie Le Corre, Mathieu Siol, Yves Vigouroux, Maud Tenaillon, Christophe

Délye

To cite this version:

Valérie Le Corre, Mathieu Siol, Yves Vigouroux, Maud Tenaillon, Christophe Délye. Adaptive

in-trogression from maize has facilitated the establishment of teosinte as a noxious weed in Europe.

Proceedings of the National Academy of Sciences of the United States of America , National Academy

of Sciences, 2020, 117 (41), pp.25618-25627. �10.1073/pnas.2006633117�. �hal-03007689�

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Main Manuscript for

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Adaptive introgression from maize has facilitated the establishment

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of teosinte as a noxious weed in Europe

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Valérie Le Correa1_{, Mathieu Siol}a_{, Yves Vigouroux}b_{, Maud I. Tenaillon}c_{, Christophe Délye}a

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a_{Agroécologie, AgroSup Dijon, INRAE, Université Bourgogne Franche-Comté, F-21000 Dijon, France}

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b_{DIADE, Université Montpellier, IRD, F-34394, Montpellier, France}

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c_{Génétique Quantitative et Evolution – Le Moulon, INRAE, Université Paris-Sud, Centre National de la}

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Recherche Scientifique, AgroParisTech, Université Paris-Saclay, F-91190, France

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1 _{Corresponding author. Email: valerie.le-corre@inrae.fr}

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Valérie Le Corre : https://orcid.org/0000-0001-6515-7795

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Mathieu Siol : https://orcid.org/0000-0003-2743-0986

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Yves Vigouroux : https://orcid.org/0000-0002-8361-6040

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Maud Tenaillon : https://orcid.org/0000-0002-0867-3678

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Christophe Délye : https://orcid.org/0000-0003-3290-3530

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Classification: BIOLOGICAL SCIENCES, EVOLUTION

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Keywords: plant invasion, rapid adaptation, genetic introgression, flowering time, herbicide

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resistance.

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Author Contributions: V.L.C, M.S, Y.V. M.I.T. and C.D. co-designed research; V.L.C., M.S. and C.D.

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performed research and analyzed data; V.L.C., M.S., M.I.T., Y.V. and C.D. wrote the manuscript and

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V.L.C. coordinated the overall study.

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Global trade has considerably accelerated biological invasions. The annual tropical teosintes, maize

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closest wild relatives, were recently reported as new agricultural weeds in two European countries,

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Spain and France. Their prompt settlement under climatic conditions differing drastically from that of

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their native range indicates rapid genetic evolution. We performed a phenotypic comparison of French

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and Mexican teosintes under European conditions, and showed that only the former could complete

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their life cycle during maize cropping season. To test the hypothesis that crop-to-wild introgression

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triggered such rapid adaptation, we used single nucleotide polymorphisms to characterize patterns of

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genetic variation in French, Spanish and Mexican teosintes as well as in maize germplasm. We showed

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that both Spanish and French teosintes originated from Zea mays ssp. mexicana race “Chalco”, a weedy

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teosinte from the Mexican highlands. However, introduced teosintes differed markedly from their

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Mexican source by elevated levels of genetic introgression from the high latitude Dent maize grown in

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Europe. We identified a clear signature of divergent selection in a region of chromosome 8 introgressed

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from maize and encompassing ZCN8, a major flowering time gene associated with adaptation to high

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latitudes. Moreover, herbicide assays and sequencing revealed that French teosintes have acquired

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herbicide resistance via the introgression of a mutant target gene (ACC1) present in

herbicide-41

resistant maize cultivars. Altogether, our results demonstrate that adaptive crop-to-wild introgression

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has triggered both rapid adaptation to a new climatic niche and acquisition of herbicide resistance,

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thereby fostering the establishment of an emerging noxious weed.

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Significance Statement

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The emergence of noxious weeds poses serious threat to agricultural production. Understanding their

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origin and evolution is therefore of major importance. Here we analyzed the intriguing case of teosinte,

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a wild relative of maize originating from Mexico that recently emerged as an invasive weed in maize

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fields in Europe. Patterns of genetic variation revealed extensive genetic introgression from maize

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adapted to temperate latitudes into European teosintes. Introgressed genomic regions harbored a key

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flowering time gene and an herbicide resistance gene. Our results exemplify how adaptive introgression

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can foster the evolution of crop wild relatives into weeds difficult to control. Hybridization is an

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evolutionary force that should not be underestimated when forecasting invasiveness risks.

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Globalization of trade and transports has considerably accelerated the rate of dispersal of species

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outside their native range (1). In Europe, the rate of introduction of alien plants has increased

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exponentially during the last century (2). This rate is expected to further increase in all temperate regions

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of the Northern Hemisphere due to climate changes (3). Alien plants are a serious threat to native wildlife

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and its associated ecosystem services, and can have direct detrimental consequences on agriculture

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production or human health (4). Understanding the origin and establishment of invasive plants is

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therefore of major importance. This includes deciphering the dynamic of the genetic composition of

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populations associated with founding events and geographical expansion and identifying the adaptive

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genetic changes sustaining their habitat shifts (5,6). Such inferences are however challenging when

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introductions are ancient, histories of invasion complex and when admixture between multiple source

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populations has taken place (e.g. 7). It is therefore particularly valuable to access the very early

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colonization steps in recently introduced species (sensu 8).

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Among invasive species, those that colonize agricultural areas are interesting in several respects: they

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have immediate consequences on crop production sustainability; they may spread rapidly via

human-73

mediated dispersal and farming activities (9); they display a suite of specific adaptive characteristics

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also described as “the agricultural weed syndrome” (10). This syndrome includes traits such as seed

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dormancy, short life-cycle and high fecundity. Two broad categories of agricultural weeds can be

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distinguished: those that evolved from crop relatives and those that evolved from wild species unrelated

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to any crop (11). Crop-related weeds display particular mechanisms of adaptation including adaptive

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genetic introgression from the crops leading to the acquisition of mimicry traits (12, 13). Many

crop-79

related wild species are among the most problematic weeds worldwide. Well-known examples are

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weedy rice, wild sorghum species, and wild sunflower species (12, 13).

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Here we focused on the extremely recent invasion of Europe by emerging noxious weeds related to

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maize (Zea mays ssp. mays), i.e. the annual teosintes. The European Food Authority has officially

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reported the presence of teosinte as weeds in maize production areas in Spain and France in 2016 (14).

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In Spain, teosintes have invaded an area in the provinces of Aragon and Catalonia where they cause

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important yield loss in maize fields (15). In France, teosintes are present in the north of the Nouvelle

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Aquitaine region. According to a technical report, French teosintes were first observed in the early 1990’s

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(16). In their native range, teosintes most closely related to maize (i.e. from the Zea mays species)

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encompass three annual subspecies: ssp. huehuetenangensis with a narrow distribution in western

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Guatemala (17), ssp parviglumis (hereafter: parviglumis) and ssp mexicana (hereafter: mexicana) both

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encountered in Mexico, the cradle of maize domestication. Parviglumis is considered as the ancestor of

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maize (18, 19) and grows in the west coast lowlands of Mexico under warm and humid tropical

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conditions. Mexicana grows in the central highlands of Mexico, at elevations up to 2800 meters, under

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cooler and drier conditions (17). The geographical distributions of these two subspecies slightly overlap

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and hybridization occurs (20). Interestingly, gene flow from mexicana to maize has contributed to

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highland adaptation of maize landraces (21). Field observations in Mexico describe parviglumis as

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forming large populations in natural and semi-natural habitats, whereas mexicana is mainly observed

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as a weed within maize fields, where it can cause severe yield loss (22-24).

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While genetic assessment of French weedy teosintes is currently lacking, two previous studies have

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attempted to establish the origin of Spanish teosintes. Their genetic characterization though single

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nucleotide polymorphism (SNP) genotyping combined with existing SNP datasets for maize and

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Mexican teosinte populations has however failed to clearly group Spanish teosintes with either

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parviglumis or mexicana. Instead, Spanish teosintes were found intermediate between maize and

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mexicana (25). Microsatellite markers further confirmed the importance of maize contribution to the

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genetic make-up of Spanish teosintes (26). Here, we collected French teosinte populations and describe

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for the first time their genetic diversity. This new dataset was combined with previously published ones

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in order to, (i) elucidate the taxonomic origin of French teosintes and identify the source populations, (ii)

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assess their genetic similarity with Spanish teosintes, (iii) describe the extent of genetic admixture

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between European teosintes and cultivated maize and (iv) identify genomic regions that have

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contributed to the successful adaptation of teosintes as weeds in European maize fields.

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Spanish and French teosintes both originate from Zea mays ssp. mexicana

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Phenotypic data from a common garden experiment conducted in Dijon demonstrated that French

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teosintes displayed two morphological characteristics, sheath pubescence (52% of plants) and

red-117

colored sheaths (75% of plants), that were observed also in some mexicana plants but not in

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parviglumis, as previously described (20) (SI Appendix, Fig. S1). Genetic variation at 24,544 SNP data

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was first investigated using Principal Component Analysis (PCA). A clear genetic structuring between

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teosintes and maize appeared along the first axis that explained 8% of the variation (Fig. 1A). The

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second axis representing 4.6% of the variation, separated parviglumis, mexicana and the European

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teosintes into three non-overlapping groups (Fig. 1A). This second axis revealed a much closer proximity

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of European teosintes to mexicana than to parviglumis.

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Results from fastStructure (27) at the subspecies level (K=3, ssp. parviglumis, ssp. mexicana, ssp.

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mays) further supported this observation, with European teosintes being of predominant mexicana

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ancestry (Fig. 1B). Increasing the number of genetic groups to K=11 (Fig. 1B and SI Appendix, Dataset

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S1) confirmed previous reports defining, in addition to the Spanish and French teosintes, nine reference

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genetic groups (28-32): (i) parviglumis accessions clustered into four geographical genetic groups

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(hereafter, PARV1, PARV2, PARV3 and PARV4) (Fig. 1B and SI Appendix, Fig. S2); (ii) some

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parviglumis populations were highly admixed with mexicana (29); (iii) mexicana accessions grouped into

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two genetic clusters (hereafter MEX1 and MEX2) corresponding to geographical races «Chalco» and

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«Central Plateau», respectively (17, 29, Fig. 1B and SI Appendix, Fig. S2); (iv) in maize, the three

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observed genetic clusters corresponded to three major germplasm pools (Fig. 1B), the tropical

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landraces, the Dent inbred lines and the Flint inbred lines (hereafter TROP, DENT and FLINT), with

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admixture among them (31, 32). In agreement with the PCA results, the results of FastStructure at K =

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11 separated French and Spanish teosinte populations in two distinct genetic clusters, different from the

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nine reference genetic groups found across parviglumis, mexicana and maize clusters.

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Consistently with results at K=3, the average pairwise genetic differentiation FST between the

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French/Spanish teosintes with mexicana accessions (0.138/0.195) was on average smaller than with

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the parviglumis populations (0.195/0.213) (SI Appendix, Table S1). Pairwise FST between French and

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Spanish teosinte populations was 0.237, a value greater than that observed between mexicana and

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parviglumis populations (0.105). Genetic diversity within groups as measured by Nei's heterozygosity

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was similar for Spanish (0.251) and French teosintes (0.221). These values stand within the range of

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genetic diversity estimates both within mexicana (0.273 for MEX1 and 0.258 for MEX2) and within

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parviglumis clusters (ranging from 0.131 to 0.305; SI Appendix, Table S1).

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We further employed the f-statistics framework (33) to test histories of divergence among parviglumis,

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mexicana, and the European teosintes, using Zea luxurians as an outgroup. Observed values of

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f4(French or Spanish teosinte, Z. luxurians; mexicana, parviglumis) were consistently significantly

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positive, again arguing in favor of a tree topology where both the French and the Spanish teosintes

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populations are more closely related to mexicana than to parviglumis (Fig. 2A). The f4 values observed

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for the two mexicana genetic clusters, MEX1 and MEX2, were however similar, so that the origin of

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European teosinte could not be more precisely refined using this statistic.

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Footprints of admixture from maize to the European teosintes

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The FastStructure analysis detected footprints of maize admixture within French and Spanish teosintes

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(Fig. 1B). In order to examine admixture patterns in more details, we used TreeMix (34) to reconstruct

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phylogenetic relationships among the nine reference genetic groups defined by fastStructure

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(ancestry>0.8, SI Appendix, Dataset S1) and the European teosintes. Without migration, the topology

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inferred was in agreement with the known relationships among subspecies (SI Appendix, Fig. S3A).

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European teosintes were most closely related to mexicana. This topology explained 98.4% of the

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observed covariance among populations. Adding five migration events increased the proportion of

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variation explained (99.7%), with the likelihood reaching an asymptote (SI Appendix, Fig. S3C-D). In the

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maximum-likelihood tree with five migration events (Fig. 2B), both French and Spanish teosintes were

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closest to the mexicana reference group MEX1, the “Chalco” mexicana group.

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Treemix analyses pinpointed migration between Dent and Tropical maize lines, likely reflecting the

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admixed origin of Corn Belt Dents between Northern Flint ancestors and tropical material (31). There

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was also evidence for admixture between maize (ancestral node or edge) and both parviglumis and

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mexicana. Those events are well documented (18, 20-21). More importantly, both French and Spanish

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teosintes displayed admixture from the Dent maize reference group (Fig. 2B). Note that the migration

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event between Dent maize and Spanish teosintes was the most-supported with an estimated weight of

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0.39, while the migration edge between Dent maize and French teosintes was added in third (SI

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Appendix, Fig. S4, estimated weight=0.14).

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Admixture between European teosintes and each of the three maize reference group was further tested

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using a four-population test where each mexicana reference group was used as a sibling population:

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f4(European teosinte group, mexicana reference group; maize reference group, parviglumis). We first

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verified that none of the reference mexicana group was itself admixed with either maize or parviglumis

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by estimating f4(MEX1, MEX2; maize, parviglumis), which was consistently not significantly different

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from zero (SI Appendix, Table S2). The four-population tests for admixture in French or Spanish teosinte

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instead were all significant (SI Appendix, Table S2). In agreement with estimated weights for migration

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edges, Z-scores were greater for Spanish teosintes in comparison to French teosintes, and for the Dent

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maize group in comparison to Tropical and Flint. The proportion of Dent maize ancestry estimated using

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the f4 ratio estimator was 0.122 (95% confidence interval 0.114 – 0.131) for French teosintes and 0.422

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(95% confidence interval 0.411 – 0.434) for Spanish teosintes.

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Introgression from maize has contributed to the adaptation of teosintes in Europe

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As tropical, short-day plants, native teosintes flower very late or not at all at higher latitudes (35, 36). A

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shift towards long-day flowering was therefore necessary for European teosintes to adapt to temperate

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latitudes. To verify these predictions, we grew plants from all French and from 12 populations of native

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teosintes in Dijon, France. French teosintes initiated their male flowering from the end of June to the

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end of July (534 to 1059 growing degree-days after sowing). In contrast, we observed a much-delayed

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transition to flowering in native teosintes. A single plant out of 24 Mexican mexicana and a majority of

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Mexican parviglumis plants (17 out of 24) remained vegetative until the end of the experiment (at the

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beginning of November). When occurring, flowering started at the beginning of September (1703

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degree-days) in mexicana and at the end of October (2221 degree-days) in parviglumis (Fig. 3A). The

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flowering of most French teosintes overlapped with the flowering of the three European maize varieties

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used as controls (which flowered respectively at 782, 904 and 987 degree-days). Synchronous flowering

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with maize was also observed in infested fields (Fig. 3B). Altogether, these results suggested that

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flowering-time genes played a prominent role in teosinte adaptation to European day length.

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We sought signatures of selection using our SNP data, paying specific attention to flowering time genes.

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We first conducted a PCA using mexicana and European accessions. Second, we performed a genome

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scan using pcadapt (37) based on squared loadings of the two first principal components (SI Appendix,

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Fig. S5). The two first principal components together explained 15% of the variation. The first component

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captured the differentiation between native mexicana and European teosintes (Fig. 4A), and we detected

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45 outlier SNPs significantly associated with it at an FDR of 0.1% (Fig. 4B). The second component

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mainly differentiated the French teosintes from the Mexicana and Spanish teosintes (Fig. 4A) and

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revealed only 2 significant outlier SNPs (SI Appendix, Fig. S6). Twenty-five out of the 45 outliers

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detected with component 1 (>55%) were found on chromosome 8 (Fig. 4B). Interestingly, we observed

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elevated levels of genetic differentiation (FST > 0.60) of both French teosintes and Spanish teosintes

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with native mexicana at a subset of 12 outlier SNPs all located on the same region of chromosome 8

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(SI Appendix, Fig. S7).

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We hypothesized that this pattern resulted from a recent adaptive introgression of a maize chromosomal

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fragment into the European teosintes. Searching for maize introgression using the ELAI software indeed

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revealed a peak of introgression from maize to the European teosintes on chromosome 8 (Fig. 5). A

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cluster of 9 adjacent outlier SNPs distant by less than 10kb was located under this peak. Remarkably,

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this cluster included the major maize flowering time gene ZCN8 (38). Note that a second major flowering

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time gene, RAP2.7, and its regulator, VGT1 (39) were located in the introgressed region but not in close

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vicinity to any of the outlier SNPs. Patterns of linkage disequilibrium (LD) in the region surrounding these

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two candidate genes uncovered a signal of elevated LD around ZCN8 in French teosintes and to a

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lesser extent in Spanish teosintes (Fig. 5). We recovered no such pattern around RAP2.7. A detailed

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examination of genotypes along chromosome 8 further confirmed the presence of a high frequency

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extended haplotype at ZCN8 in French teosintes. This same haplotype seems present in the Spanish

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teosinte but is still segregating, suggesting weaker or partial selection at this position if any (SI Appendix,

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Fig. S8).

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Acquisition by French teosintes of an herbicide resistance gene introgressed from maize

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In France, maize cultivars resistant to the herbicide cycloxydim carry an allele of the gene encoding

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acetyl-CoA carboxylase 1 (ACC1) with a mutation that confers resistance to herbicides inhibiting this

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enzyme (Duo System®, 40). Because French teosintes grow in the vicinity of such cultivars, we

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assessed their sensitivity to cycloxydim. Out of 200 French teosinte seedlings assayed, 86 (43%) were

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rated resistant to cycloxydim and 114 (57%) were rated sensitive. As controls, we used a sensitive (RGT

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TetraXX) and a resistant (RGT CarmiDuo) maize cultivar. As expected, all plants from the former cultivar

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were rated sensitive while all plants from the latter were rated resistant. To explore the possibility that

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resistant alleles were transferred from maize to teosintes, we genotyped the ACC1 gene in teosintes

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and maize at all known codons involved in herbicide resistance. We detected one mutation at codon

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1781 (Ile to Leu) in the resistant maize variety (RGT CarmiDuo) and in some of the French teosintes.

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All CarmiDuo maize plants were homozygous mutant. All plant from the sensitive maize cultivar

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(TetraXX) were homozygous wild-type. Among the French teosinte plants assayed for herbicide

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sensitivity, all 86 herbicide-resistant plants were homozygous mutants at codon 1781. Among the 114

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herbicide-sensitive teosinte plants, 78 were heterozygous and 36 were homozygous wild-type.

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ACC1 is located on chromosome 2, in a region where a peak of squared loadings was observed with

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the second principal component of the PCA differentiating the French from the Spanish teosintes (SI

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Appendix, Fig. S6). Although it did not pass the FDR at 0.1%, this region was associated with a large

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divergence between French and Spanish teosintes (Fig. 4A). When examining the pattern of

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introgression over the chromosome 2 using ELAI, it was clear that a large genomic region encompassing

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ACC1 and spanning more than 500 SNPs from our genotyping chip had been introgressed from maize

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into homozygous ACC1-mutant teosinte plants, whereas wild-type plants showed no introgression and

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heterozygotes showed the expected halved ancestry dosage (Fig. 6A). Sequencing the full genomic

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DNA corresponding to the coding sequence of ACC1 revealed that all mutant teosintes carried one

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same ACC1 allele that was exactly identical to the allele carried by herbicide-resistant maize cultivars,

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whereas wild-type ACC1 alleles carried by non-mutant French teosintes clustered with the alleles carried

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by the mexicana accessions (Fig. 6B). Taken together, these results demonstrate that the mutant ACC1

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allele present in French teosinte was introgressed from cultivated, herbicide-resistant Duo System

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We report here the first genetic description of the very recent settlement in France of teosintes, which

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are recognized in their native tropical range as a major threat to agricultural production (41). We

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addressed three main questions: Where do they originate? How did they adapt to Europe? To which

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extent has introgression from maize facilitated their establishment?

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Our results clearly assigned both French and Spanish teosintes (25) to Zea mays ssp. mexicana, and

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suggested a single geographical origin for all invasive populations of teosintes reported to date in

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Europe. We identified the source genetic group as the mexicana race “Chalco” (MEX1). This finding is

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interesting in at least two respects: first, Chalco teosintes are located at elevations ∼2300 m and higher

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(29). They are therefore adapted to moderate rainfall and low temperatures (17), environmental

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conditions that are closer to the European climate than the Mexican tropical lowlands. Second, Chalco

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teosintes are the ones that hybridize the most frequently with maize (42, 43). Plants carrying hybrid-like

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cobs have been reported frequently in Mexico for Chalco teosintes growing within or near maize fields

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(23, 24). In fact, Chalco teosintes have consistently been described as weeds infesting cultivated maize

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fields (22, 24, 41). This parallels the sites colonized in France and Spain, which are chiefly maize fields

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(15).

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However, the native and introduced ranges differ strongly in their latitude and hence their photoperiod,

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short-days in the native range versus long-days in the introduced one. In Mexico, mexicana occurs at

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latitudes comprised roughly between 18° North and 20° North. In Europe, invasive mexicana are

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observed at latitudes comprised between 42° North (in Spain) and 46° North (in France), which would

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correspond to an area north of Chicago in the US. Flowering is accelerated by short-days in native

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teosintes (35). We indeed confirmed that native mexicana populations flowered very late in France and

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were unable to produce seeds before maize harvest time. On the contrary, the flowering period of French

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teosintes was much earlier, and overlapped that of maize. The establishment of mexicana in Europe

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therefore most likely involved a substantial genetic shift in the control of flowering time.

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Given the narrow time window for adaptation to occur from de novo mutations (two to three decades),

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we combined outlier detection in European teosintes and the examination of introgression patterns from

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maize to test whether pre-adapted local maize varieties had contributed to teosinte adaptation. In line

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with this hypothesis, we detected introgression in both Spanish and French teosintes. Interestingly, we

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observed a marked pattern of introgression in a genomic region that contains ZCN8, a gene that

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underlies one of the largest maize flowering time quantitative trait locus (QTL). Consistently, this region

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was enriched for outlier SNPs displaying high differentiation between native and European mexicana

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populations. ZCN8 is a key floral activator of the maize flowering time pathway also known to be involved

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in photoperiod sensitivity (44). Guo et al. (38) have shown that two polymorphisms with an additive effect

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in the promoter of ZCN8 are associated with early flowering time under long days. These polymorphisms

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exist as standing variation in mexicana. They have been under strong selection during early maize

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domestication and contributed to latitudinal adaptation in this crop (31, 38). While introduction to Europe

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of a pre-adapted, early-flowering mexicana population is a possibility, here we propose that introgression

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from a maize early flowering variant at ZCN8 opened up a new niche for weedy teosintes in Europe.

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Indeed, a clear signal of selective sweep was observed around ZCN8 in French teosintes, consistent

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with a single event of adaptive introgression, i.e hard sweep signature. Interestingly, a similar haplotype

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was observed in Spanish teosintes, albeit with a greater level of heterozygosity, which suggests an

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ongoing, incomplete selective sweep (SI Appendix, Figure S8). Genetic introgression was not limited to

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this genomic region but was pervasive in all European teosintes (SI Appendix, Fig. S9). Given the

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complexity and number of genes involved in the regulation of maize flowering time, we suspect several

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genes other than ZCN8 to contribute to the substantial shift in flowering time in European teosintes (see

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SI Appendix, Fig. S9 and Dataset S2 for a proposed list of candidate genes). Our experimental design,

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however, recovered no specific selection signal at any of the a priori flowering time candidate genes.

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We pinpointed the Dent genetic group as the most likely donor of the introgressed segments. Because

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modern maize varieties deriving from the Dent germplasm are widely cultivated in Europe but not in

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Mexico, where tropical germplasm is dominant (32; 45), this suggests that hybridization has occurred

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after the introduction of mexicana to Europe. In other words, our results are consistent with a scenario

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where European maize varieties adapted to temperate latitudes have contributed to the establishment

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of teosintes in Europe. Note that hybridization is seemingly still occurring, as plants carrying hybrid-like

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cobs are regularly observed in infested maize fields in Spain (25, 26) as well as in France (SI Appendix,

309

Fig. S10). One surprising outcome of our study is the much lower global introgression rate in the French

310

as compared to the Spanish teosintes (SI Appendix, Fig. S9), despite the seemingly earlier French

311

introduction. Introgression segments can convey alleles that are deleterious for the maintenance of

312

weediness traits and eliminated over time by purifying selection (13), perhaps explaining subtler

313

introgression patterns in French teosintes. Along this line, we detected very little introgression from

314

maize to European teosintes on an extended portion of the short arm of chromosome 4 (SI Appendix,

10

Fig. S9). This region encompasses a large domestication QTL hotspot containing several loci involved

316

in the variation of domestication traits between maize and teosinte, as well as the incompatibility locus

317

TCB1 (46, 47, see SI Appendix, Fig. S9 and Dataset S2). Our results, together with those of a previous

318

study (43) strongly suggest that introgression from maize to mexicana is counter-selected in this

319

genomic region. In European teosintes, such selection most likely contributes to preserve wild alleles

320

necessary for the maintenance of the weedy phenotype.

321

Last, but not least, we detected a second adaptive introgression specific to the French teosintes and

322

involving a large region of chromosome 2. This region encompasses an allele of the herbicide-target

323

gene ACC1 carried by herbicide-resistant maize cultivars. Such cultivars have been authorized in

324

France since 2001 and are cultivated in the area where teosintes occur (16). Teosinte plants

325

homozygous for this mutant allele are herbicide-resistant, which is clearly an adaptive trait in agricultural

326

fields. Since the history of cultivation of resistant maize cultivars in France is quite recent, it follows that

327

the introgression of the ACC1 region in French teosintes is also very recent, consistent with the fact that

328

a large region of chromosome 2 encompassing ACC1 was introgressed.

329

In conclusion, while introgression has been proposed as a key source of adaptive genetic variation (48,

330

49), establishing it has been particularly challenging with only a handful of reported cases. Notorious

331

examples often illustrate the contribution of wild relatives to domesticated gene pools (50), more rarely

332

the reverse (but see 51-53). This is because crop-to-wild gene flow may not always be beneficial since

333

many characteristics making crops suitable to cultivated environments are detrimental for wild or weedy

334

forms (e.g., non-shattering seeds, lack of dormancy, bolting time) (10). In some instances, however, a

335

crop allele can provide a clear advantage to a wild or weedy form, e.g. by conferring a given resistance

336

or by allowing a niche shift. Here, we present two clear evidence of adaptive introgression from locally

337

adapted crop varieties to a wild relative. One introgression facilitated reproduction under temperate

338

latitudes, the other enabled plants to thrive in herbicide-treated fields. Together, those introgressions

339

contributed to the establishment of a new weed. Previous studies have reported a low rate of

340

spontaneous hybridization between mexicana and cultivated maize, less than 1% per generation (54).

341

However, first-generation hybrids display a great vigor, and are highly male fertile (55). We propose that

342

the rare first-generation hybrids served as a bridge for the transfer of maize genes into mexicana

343

populations, fostering their local adaptation. This result nicely parallels the contribution of mexicana

344

alleles to highland maize landraces adaptation (42). In sum, we demonstrate that crop-wild introgression

345

can be a two-way street, allowing the transfer of beneficial variants to both partners. Our work highlights

346

the importance of introgression in allowing large evolutionary shifts or even opening up new niches. In

347

the case of maize and teosinte, the common consensus was that given their ecology and biology, the

348

risk of seeing teosinte emerge as a problematic weed under a temperate climate was remote (17, 20).

349

Here we not only show that such risk exists, but more generally that crop-wild introgression should not

350

be underestimated when forecasting invasiveness risks.

351

352

11

353

Materials and Methods

354

355

Plant material and genotyping

356

Teosinte seeds were collected from eight cultivated fields in the region of Nouvelle Aquitaine, France,

357

in autumn 2017 (SI Appendix, Table S3). Geographic distances between fields varied from 0.25

358

kilometers to 11 kilometers. In spring 2018, seeds were germinated in growth chambers at 25°C and

359

16h day length. Leaf samples were harvested from a total of 70 French teosintes individuals (4 to 14

360

individuals per field population). Leaf fragments were ground in liquid nitrogen and DNA extracted using

361

kit NucleoSpin Plant II (Macherey-Nagel). Genotyping was performed by Eurofins Genomics using the

362

Illumina MaizeSNP50 BeadChip array (Illumina, San Diego, CA, USA). SNPs were called using the

363

GenomeStudio algorithm (Illumina). Out of 56,110 markers contained on the chip, 49,574 could be

364

successfully genotyped on all plants.

365

Phenotypic assays

366

A common garden experiment was conducted in 2018 at INRA in Dijon, France (47.32°N; 05.10°E) to

367

compare phenotypic variation in French and Mexican teosintes. Seed material from six populations of

368

the subspecies mexicana and six populations of the subspecies parviglumis were obtained from M.I.

369

Tenaillon (collection described in 56, 57). This material was used as reference material in the common

370

garden (see below).The experiment included 48 plants from the eight populations of French teosinte

371

(six plants per population), 24 plants from the six mexicana populations (three to five plants per

372

population) and 24 plants from the six parviglumis populations (four plants per population). We included

373

three maize varieties commercialized in France: ES Gallery (36 plants), RGT CarmiDuo (12 plants) and

374

RGT TetraXX (12 plants). All seedlings were transplanted one week after sowing and arranged in a

375

semi-randomized single block design with alternate rows of teosinte and maize. The experiment was

376

set up on the 24th _{of May and ended on the 6}th _{of November. We measured traits related to early growth}

377

and architecture (plant height, number of leaves on the main tiller and number of primary tillers), leaf

378

shape (length, width and their ratio), the presence of trichomes on leaf sheaths, sheath color and

379

flowering time (time to emission of the tassel and time to silking). Due to the much-delayed transition to

380

reproduction in Mexican teosintes (see results), post-reproductive traits were not considered.

381

Herbicide sensitivity bioassay

382

French teosintes have almost exclusively been observed in maize-growing fields. Growers in the area

383

where teosintes are present have tried to control it using non-genetically modified, herbicide-resistant

384

maize cultivars (Duo System®, BASF) that withstand the application of the herbicide cycloxydim.

385

Bioassays were conducted to assess the herbicide sensitivity of French teosinte seedlings issued from

386

seeds from the eight populations collected in maize fields. Seedlings were grown in individual pots in a

387

glasshouse at 22/18 °C day/night with 14-h photoperiod. At the 2-leaf stage, cycloxydim was applied as

12

the commercial herbicide Stratos Ultra (BASF, 100 g/L cycloxydim) at the recommended French field

389

rate (200 g/ha cycloxydim) on 200 teosinte seedlings (25 per population) and on 25 seedlings from each

390

of one classical (RGT TetraXX) and one herbicide-resistant (RGT CarmiDuo) maize cultivar that were

391

included as herbicide-sensitive and herbicide-resistant controls, respectively. Twenty-five additional

392

French teosinte seedlings and 25 seedlings of each maize cultivar were sprayed with water to serve as

393

an untreated control. After 48 hours, the last 0.5 cm of the first leaf of every sprayed seedling was

394

collected for ACCase genotyping (see below). Plants phenotypes were rated three weeks after herbicide

395

application, when herbicide-sensitive control maize plants were clearly dead. Plants killed by the

396

herbicide were rated sensitive (S), while surviving plants were rated resistant (R).

397

SNP array data

398

Genotype data for the 70 French teosintes was combined with published and available data for the

399

following material: 40 accessions of Spanish teosintes (25), 314 accessions of parviglumis (28, 29), 332

400

accessions of mexicana (28, 29), 94 maize landraces from Meso- and Central-America (58) and 155

401

maize inbred lines from North-America and Europe (32). We only kept SNPs that were shared and

402

correctly scored among the different datasets, the final combined dataset consisted of 24,544 SNPs

403

genotyped on 1,005 accessions (http://doi.org/10.5281/zenodo.3959138). For analyses requiring an

404

outgroup, we included the SNPs data available for twelve accessions of Zea luxurians (25) using the

405

24,544 markers above.

406

Acetyl-CoA carboxylase genotyping and sequencing

407

Herbicide-resistant, Duo system maize cultivars grown in French fields to control teosinte populations

408

all carry an herbicide-resistant mutant allele of ACC1, one of the two maize acetyl-CoA carboxylases

409

(40). The mutation involved has not been published, but the major acetyl-CoA carboxylase codons

410

involved in herbicide resistance are known (codons 1781, 1999, 2027, 2041, 2078, 2088 and 2096 as

411

standardized in 59). Two herbicide-resistant maize cultivars (RGT CarmiDuo and RGT EXXplicit) and

412

the 200 French teosinte plants used in herbicide sensitivity bioassays were genotyped at these codons.

413

The sequences of the two maize acetyl-CoA carboxylases homeologs (Genbank accessions

414

XM_020548014 for ACC1 and XM_008664827 for ACC2) were aligned and gene-specific primers were

415

designed for ACC1. Primers pairs AC1ZM3/AC1ZM3R and AC1ZM2/AC1ZM2R (SI Appendix, Table

416

S4) were used to amplify ACC1 regions carrying codon 1781 and codons 1999 to 2096, respectively.

417

Mutations were sought in the amplicons obtained using previously described assays (59).

418

The ACC1 protein-coding sequence of 12,002 nucleotide with its 32 introns was fully sequenced on both

419

strands in 14 individual plants: one plant from each of the two herbicide-resistant maize cultivars, three

420

French teosinte individuals homozygous mutant at ACC1 and three homozygous wild-type at ACC1 as

421

determined after genotyping, one parviglumis individual in each of two Mexican populations, one

422

mexicana individual in each of two Mexican populations, and two Zea mays ssp. huehuetenangensis

423

individuals that were used as an outgroup. PCR primers used for sequencing are in SI Appendix, Table

13

S4. All sequences were aligned with the maize reference ACC1 sequence (genbank XM_020548014).

425

A phylogenetic tree was generated using the Neigbour-joining method as implemented in Mega 10.0.5

426

(60) with 1,000 bootstraps.

427

Population genetic structure

428

A principal component analysis (PCA) was conducted using the Adegenet R package (61). The

429

clustering program FastStructure (27) was run to evaluate ancestry proportions for K genetic groups,

430

with K varying from 1 to 12 with five replicates for each value of K and using the “simple prior” option

431

(flat beta-prior over allele frequencies). To evaluate the repeatability across runs, and rule out true

432

multimodality (as opposed to cluster labels switching), we ran the program CLUMPP v.1.1.2 using the

433

Greedy algorithm (62). Genetic diversity within each genetic group and pairwise genetic differentiation

434

(FST) values were calculated using the last version of the EggLib package (63).

435

Origin of European teosintes

436

In this analysis, we aimed at inferring the Mexican origin of European teosintes. We first defined Mexican

437

reference groups of parviglumis and mexicana. We considered the results from FastStructure at K=11,

438

as this was the value for which the observed genetic clustering for Mexican teosintes and maize was in

439

best agreement with previous studies (28-32). This clustering revealed six teosinte genetic groups (four

440

from parviglumis, two from Mexicana) as well as three maize genetic groups (tropical landraces, Dent

441

and Flint inbred lines). We retained individuals with an ancestry higher than 0.8 in each group. This set

442

of 628 individuals defined our nine reference groups (SI Appendix, Dataset S1).

443

To get a first insight on the proximity between each European teosinte population (Spanish and French)

444

and the reference groups of parviglumis and mexicana, we used the f-statistics first introduced by Reich

445

(64). F-statistics provide a measure of genetic drift between populations, based on the branch length

446

separating them on a simple phylogeny (65). The four-population f4-statistics can be used to investigate

447

ancestry relationships and find the closest relative of a contemporary population by comparing different

448

tree topologies (33; 19). We used f4 (European teosinte, Zea luxurians; mexicana, parviglumis), where

449

mexicana and parviglumis are the two putative ancestors to European teosintes and Zea luxurians is an

450

outgroup. The value of this f4 statistics is expected to be positive if the European teosinte descends

451

from mexicana, negative if it descends from parviglumis and null in case of no ancestry relationship (see

452

19 for a similar analysis). Observed f4 values were calculated using the fourpop program in TreeMix

453

1.13 (34). Note that we make here the implicit assumption of no gene flow between reference groups.

454

We considered more complex scenarios in the following section.

455

History of admixture among teosintes and maize

456

We inferred the relationship between cultivated maize and teosintes using Treemix 1.13 (34) on the nine

457

reference genetic groups defined above. The analysis was based on SNPs allele frequencies in Spanish

458

teosintes, French teosintes and the nine reference genetic groups. Maximum likelihood trees were built

14

using 200 SNP-windows to account for linkage disequilibrium. We tested the addition of 0 to 10 migration

460

events, by building 10 replicate trees for each. We considered as the most meaningful number of

461

migration events the first value at which the mean likelihood of trees and the proportion of explained

462

covariance among groups stabilized towards their maximum asymptotic values.

463

As both the comparison of f-statistics for varying tree topologies and Treemix results assigned mexicana

464

as the most likely ancestor of European teosintes, we performed a four-population test (64, 65)

465

considering (test population, mexicana; maize, parviglumis). We estimated the f4 statistics for all

466

combinations of the test population being either Spanish or French teosintes, and considering the two

467

mexicana reference groups (MEX1, MEX2), the three maize reference groups, and all parviglumis

468

reference groups grouped together. The expected value of this f4 statistics is zero if (test population,

469

mexicana) and (maize, parviglumis) form two independently diverged clades. Significant deviation from

470

zero indicates admixture. Before implementing this test, we verified that the two reference mexicana

471

groups were not themselves admixed with either maize or parviglumis, which would confuse

472

interpretation. We did so by estimating f4 (MEX1, MEX2; maize, parviglumis). Finally, under an

473

admixture scenario of the test population with maize, the admixture proportion in the test population was

474

estimated as the ratio of the two statistics f4 (parviglumis, Zea luxurians; test population, mexicana) and

475

f4 (parviglumis, Zea luxurians; maize, mexicana). Here, in line with the reasoning in Patterson et al. (65)

476

and Peter (33), we used Zea perennis as the outgroup, mexicana and maize as the two potential

477

contributors to the admixed test population, and parviglumis as a subspecies more closely related to

478

one of the contributors, here to maize. A 95% confidence interval for the admixture proportion was

479

obtained from a block jackknife procedure, where each block of 200 SNPs was removed in turn.

480

Signatures of selection and genomic patterns of introgression

481

A genome-wide scan for signature of positive selection in European teosintes was performed using a

482

principal component analysis over all European teosintes and mexicana populations as implemented in

483

pcadapt (64). In contrast to FST-based approaches, pcadapt does not require any a priori grouping of

484

individuals into populations. It is well suited to scenarios of population divergence and range expansion,

485

as principal components are able to discriminate successive divergence and selection events (37). We

486

performed the analysis for each principal component (component-wise method) and used the loadings

487

(correlation between each PC and each SNP) as the test statistic. Outlier SNPs were identified by

488

transforming the p-values into q-values with a cut-off value of 0.001, ensuring a false discovery rate

489

lower than 0.1% using the R package qvalue (67).

490

We investigated genome-wide patterns of introgression from cultivated maize using the ELAI software

491

(68). Parameters used were 2 upper-layer clusters and 10 lower-level clusters, 30 EM steps and 10

492

generations of admixture between the two source populations identified in the Treemix and f-statistics

493

analyses (non-admixed reference genetic groups as identified above, namely MEX1 and DENT). We

494

thus analyzed each French (Spanish, respectively) teosinte individual as resulting from the introgression

495

between the haplotypes of the two sources populations, MEX1 and Dent. We then plotted the average

15

ancestral allele dosage over all French (Spanish, respectively) teosinte individuals.ELAI analyses were

497

performed separately for each chromosome.

498

499

500

Acknowledgments and funding sources

501

502

We thank Bruno Chauvel (INRAE) for bringing to our attention the presence of teosintes in maize fields

503

in France. We thank Séverine Michel from INRAE for herbicide sensitivity bioassays and molecular

504

analysis of the ACC1 gene. We thank Delphine Madur from GQE-Le Moulon for handling DNA samples

505

used for SNP array genotyping. GQE-Le Moulon benefits from the support of Saclay Plant

Sciences-506

SPS (ANR-17-EUR-0007). M. I. T. and Y.V. are supported by an ANR grant (ANR-19-CE32-0009).

507

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